DESCRIPTION (Verbatim from the Applicant's Abstract): Our laboratory is interested in various aspects of the Na,K-ATPase, including structure-function relationships and understanding the regulated expression of the isoforms of the alpha and beta subunits. We are especially interested in the physiological role this enzyme plays and have recently developed animals lacking the alpha1 and alpha2 isoforms. Of particular significance from these studies is the finding that each isoform plays a different role in heart and skeletal muscle contraction. We wish to follow up on these studies as well as pursue others that are related to the in vivo role of this enzyme. The specific aims of our grant are to use a replacement gene targeting approach to determine whether the cardiac glycoside binding site of the Na,K-ATPase plays a biological role. Such studies will help define whether natural ligands exist for this enzyme and the role that they might play. In our second aim, we will use these animals to define the role of individual alpha isoforms in mediating the positive and negative effects of cardiac glycoside therapy. A third aim relates to understanding the physiological role of each of the alpha isoforms using chimeric analysis and tissue-specific knockouts. Animals lacking the alpha2 isoform fail to survive past birth and these animals provide an opportunity for understanding the specific biological niche that this isoform plays. In order to more fully define the tissue responsile for the lethal phenotype, we plan to carry out chimeric studies with embryonic stem cells which lack both copies of the alpha2 isoform and to carry out tissue-specific knockouts. These approaches will help define which organ system(s) is responsible for the failure of the animals to develop past birth. These studies will also likely provide animals with tissues lacking the alpha2 isoform and make it possible to define the role of this isoform in that tissue.